TEMPERATURE-SENSITIVE MUTANTS FOR THE REPLICATION OF PLASMIDS IN ESCHERICHIA COLI II. PROPERTIES OF HOST AND PLASMID MUTATIONS

David T. Kingsbury ¹, Donna G. Sieckmann ¹, and Donald R. Helinski ¹

¹ Department of Biology, University of California, San Diego, La Jolla, California 92037

Host mutations in Escherichia coli K12 selected for the temperature-sensitive replication of the bacterial plasmid colicinogenic factor E₁ (ColE₁) exhibit a pleiotropic effect with respect to the effect of the mutation on other extra-chromosomal elements. The mutations also vary with respect to the time of incubation of the cells at 43°C required for complete cessation of ColE₁ DNA synthesis. While the synthesis of the bacterial chromosome appears unaffected, supercoiled ColE₁ DNA replication stops immediately in some mutants and gradually decreases during several generations of cell growth before stopping in others. Mutations isolated in the ColE₁ plasmid resulted in only a gradual cessation of ColE₁ DNA synthesis over several generations of cell growth at 43°C. Conjugal transfer of the ColE₁ and ColV factors occurs normally in the host mutants when the transfer is carried out at the permissive temperature; however, the presence of a group I mutation in the donor cell prohibited conjugal transfer of either plasmid DNA at 43°C to a normal recipient cell. Similarly, the presence of this mutation in the recipient prevented the establishment of ColE₁ or ColV in the mutant recipient cell upon conjugation with a normal donor at 43°C. Various host ColE₁ replication mutants carrying either ColE₁ or ColE₂ were also defective in the mitomycin C-induced production of colicin E₁ or colicin E₂ at 43°C. The majority of the host mutations examined exhibited a temperature sensitivity to growth in deoxycholate in addition to the inhibition of plasmid DNA replication, suggesting a membrane alteration in these mutants when grown at the restrictive temperature.

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